Tuning Enzyme Thermostability via Computationally Guided Covalent Stapling and Structural Basis of Enhanced Stabilization.

Enhancing the thermostability of enzymes without impacting their catalytic function represents an important yet challenging goal in protein engineering and biocatalysis. We recently introduced a novel method for enzyme thermostabilization that relies on the computationally guided installation of genetically encoded thioether "staples" into a protein via cysteine alkylation with the noncanonical amino acid O-2-bromoethyl tyrosine (O2beY). Here, we demonstrate the functionality of an expanded set of electrophilic amino acids featuring chloroacetamido, acrylamido, and vinylsulfonamido side-chain groups for protein stapling using this strategy. Using a myoglobin-based cyclopropanase as a model enzyme, our studies show that covalent stapling with p-chloroacetamido-phenylalanine (pCaaF) provides higher stapling efficiency and enhanced stability (thermodynamic and kinetic) compared to the other stapled variants and the parent protein. Interestingly, molecular simulations of conformational flexibility of the cross-links show that the pCaaF staple allows fewer energetically feasible conformers than the other staples, and this property may be a broader indicator of stability enhancement. Using this strategy, pCaaF-stapled variants with significantly enhanced stability against thermal denaturation (ΔTm' = +27 °C) and temperature-induced heme loss (ΔT50 = +30 °C) were obtained while maintaining high levels of catalytic activity and stereoselectivity. Crystallographic analyses of singly and doubly stapled variants provide key insights into the structural basis for stabilization, which includes both direct interactions of the staples with protein residues and indirect interactions through adjacent residues involved in heme binding. This work expands the toolbox of protein stapling strategies available for protein stabilization.

MOrPH-PhD: A Phage Display System for the Functional Selection of Genetically Encoded Macrocyclic Peptides.

Macrocyclic peptides represent promising scaffolds for targeting biomolecules with high affinity and selectivity, making methods for the diversification and functional selection of these macrocycles highly valuable for drug discovery purposes. We recently reported a novel phage display platform (called MOrPH-PhD) for the creation and functional exploration of combinatorial libraries of genetically encoded cyclic peptides. In this system, spontaneous, posttranslational peptide cyclization by means of a cysteine-reactive non-canonical amino acid is integrated with M13 bacteriophage display, enabling the creation of genetically encoded macrocyclic peptide libraries displayed on phage particles. Using this system, it is possible to rapidly generate and screen large libraries of phage-displayed macrocyclic peptides (up to 108 to 1010 members) in order to identify high-affinity binders of a target protein of interest. Herein, we describe step-by-step protocols for the production of MOrPH-PhD libraries, the screening of these libraries against an immobilized protein target, and the isolation and characterization of functional macrocyclic peptides from these genetically encoded libraries.

Expanded toolbox for directing the biosynthesis of macrocyclic peptides in bacterial cells.

The macrocyclization of recombinant polypeptides by means of genetically encodable non-canonical amino acids has recently provided an attractive strategy for the screening and discovery of macrocyclic peptide inhibitors of protein-protein interactions. Here, we report the development of an expanded suite of electrophilic unnatural amino acids (eUAAs) useful for directing the biosynthesis of genetically encoded thioether-bridged macrocyclic peptides in bacterial cells (E. coli). These reagents are shown to provide efficient access to a broad range of macrocyclic peptide scaffolds spanning from 2 to 20 amino acid residues, with the different eUAAs offering complementary reactivity profiles toward mediating short- vs. long-range macrocyclizations. Swapping of the eUAA cyclization module in a cyclopeptide inhibitor of streptavidin and Keap1 led to compounds with markedly distinct binding affinity toward the respective target proteins, highlighting the effectiveness of this strategy toward tuning the structural and functional properties of bioactive macrocyclic peptides. The peptide cyclization strategies reported here expand opportunities for the combinatorial biosynthesis of natural product-like peptide macrocycles in bacterial cells or in combination with display platforms toward the discovery of selective agents capable of targeting proteins and protein-mediated interactions.

MOrPH-PhD: An Integrated Phage Display Platform for the Discovery of Functional Genetically Encoded Peptide Macrocycles.

Macrocyclic peptides represent attractive scaffolds for targeting protein-protein interactions, making methods for the diversification and functional selection of these molecules highly valuable for molecular discovery purposes. Here, we report the development of a novel strategy for the generation and high-throughput screening of combinatorial libraries of macrocyclic peptides constrained by a nonreducible thioether bridge. In this system, spontaneous, posttranslational peptide cyclization by means of a cysteine-reactive noncanonical amino acid was integrated with M13 bacteriophage display, enabling the creation of genetically encoded macrocyclic peptide libraries displayed on phage particles. This platform, named MOrPH-PhD, was successfully applied to produce and screen 105- to 108-member libraries of peptide macrocycles against three different protein targets, resulting in the discovery of a high-affinity binder for streptavidin (K D: 20 nM) and potent inhibitors of the therapeutically relevant proteins Kelch-like ECH-associated protein 1 (K D: 40 nM) and Sonic Hedgehog (K D: 550 nM). This work introduces and validates an efficient and general platform for the discovery and evolution of functional, conformationally constrained macrocyclic peptides useful for targeting proteins and protein-mediated interactions.